Project Summary/Abstract The abnormal expression of cell surface receptors on tumor cells has become a major focus of efforts to individualize cancer therapy. Receptors involved in cell proliferation and programmed death are commonly targeted with antibody therapies, and new treatment modalities seek to exploit this abnormality to preferentially deliver toxic payloads to tumor cells. However, because of the complexity of tumor vasculature and leakage, the ability to noninvasively quantify the availability of these receptors remains elusive, precluding quantitative characterization and longitudinal monitoring of tumor receptor concentration. This project aims to advance a novel noninvasive MRI-coupled optical imaging approach that accounts for contrast agent pharmacokinetics and thus is capable of quantifying receptor concentration and availability in sub-surface tumors. This capability is enabled by imaging the kinetics of two fluorescent tracers injected simultaneously, one targeted to the receptor of interest, and the other to a non-targeted counterpart. Fitting the time course data to a dual-tracer compartmental model allows the recovery the density of cellular receptors available for binding. Accessing this parameter noninvasively could have a profound impact on drug development programs and even clinical practice, enabling characterization and tracking of drug targets in tumors. The aims in this project are designed to advance and validate all aspects of this technology. Specifically, a novel, low cost multispectral fluorescence tomography system dedicated to MRI-guided dual tracer fluorescence tomography (MRg-DTFT) in preclinical MRI research scanners will be developed, validated and used to explore the molecular response to new cancer therapies. Imaging performance of the instrument will be assessed using multi-tracer phantoms and by comparing in vivo animal images to co-registered fluorescence imaging of ex-vivo tissue slices. Significant development effort will also be directed towards optimizing the image processing and reconstruction algorithms required for fully integrated MRI-optical image recovery and analysis for the dual-tracer approach, with the ultimate aim of enabling one-click parameter recovery and visualization. These tools will be deployed to further validate the dual-tracer approach through extensive animal experiments which examine receptor density in multiple orthotopic glioblastoma tumors known to have varying receptor expression profiles and vascular structure. Finally, this unique capability will be deployed to investigate the effect an emerging tumor- penetrating therapeutic adjuvant has on available receptor density.